Innovare International Journal of Pharmacy and Pharmaceutical Sciences

Academic Sciences ISSN- 0975-1491 Vol 6, Issue 8, 2014

Original Article COMPARATIVE ANALYSIS OF CARDIAC GLYCOSIDES AND QUANTIFICATION OF PERUVOSIDE FROM THEVETIA NERIIFOLIA, JUSS FRUIT RIND EXTRACTS THROUGH HPTLC FINGERPRINTING

NESY E. A.,1 LIZZY MATHEW 2 1Department of Botany, K K T M Govt College, Kodungallur, Trichur, Kerala, India, 2Department of Botany, St.Teresa’s College, Ernakulam, Kerala, India. Email: [email protected] Received: 04 Jul 2014 Revised and Accepted: 05 Aug 2014 ABSTRACT Objective: Present investigation was carried out to determine the cardiac glycoside profile and to quantify the powerful cardiotonic drug peruvoside in the fruit wall of Thevetia neriifolia, Juss a medicinal plant used in indigenous system of medicine since decades. Methods: Shade dried powdered samples were subjected to soxhlet extraction using different solvents, and various forms of cardiac glycosides were analysed in all fractions using TLC plates. Positive samples for peruvoside were subjected to HPTLC fingerprinting on silica gel 60 F254 plates using chloroform: methanol (8:2v/v) mixture as mobile phase. Results: Fingerprint profile showed the presence of 6-14 cardiac glycosides in all three morphovariat plants. Densitometric analysis at 220 nm showed a peak at Rf 0.67±0.01 to the reference drug peruvoside, and in all samples analysed an identical compound corresponding to peruvoside was eluted within a range of 0.63 to 0.75 with minor variations. It was noticed that peruvoside was the third largest cardiac glycoside extracted from the fruit rind using chloroform. Conclusion: In all studied morphoforms, about 3.25% peruvoside was detected in the rind which was the neglected and unused portion of fruit. Using fingerprinting technique, all cardiac glycosides can be effectively separated and utilized for pharmaceutical purposes. Keywords: Cardiac glycosides, Fruit rind, HPTLC fingerprinting, Morphoform, Peruvoside.

INTRODUCTION rots away easily from fallen fruits. Seeds are reported to be a store house of various cardiac glycosides, however, phytochemical studies Cardiac glycosides are potent group of naturally occurring drugs and related to fruit rind remains scanty. Hence, the present investigation their distribution in plants is restricted to a few families and genera. was carried out to categorize various phytochemicals, mainly Among angiosperms, these secondary metabolites are mostly cardiac glycosides in the fruit wall of all three morpho-variants and concentrated within the members of Apocynaceae, to quantify a potent cardiotonic drug peruvoside in light of the Scrophulariaceae, Asclepiadaceae and Liliaceae. Plants use these previous documentations regarding the therapeutic significance of compounds for defense purposes against herbivores [1]. The peruvoside and other cardiac glycosides. extensive use of digitalis related glycosides for treating heart failure are well accepted from centuries back, but narrow therapeutic index MATERIALS AND METHODS between effective and toxic dozes makes their use more vigilant [2]. The term cardiac glycoside was derived from the Greek word Mature fruits of Thevetia neriifolia were collected from various fields of Trichur district, Kerala. The authenticated voucher specimens ‘kardia’, meaning heart. (2458a-c) were kept in the Herbarium cabinet of Botany A great majority of drugs are either naturally derived or synthesized Department, St. Teresa’s College, Ernakulam. Ripened fruits were from plant related compounds. Thevetia neriifolia, Juss. (T. collected, washed carefully and kept in shade for 2-3 days for the peruviana), a popular medicinal plant belongs to family ease of separation of fleshy mesocarp from stony endocarp. Pulpy Apocynaceae, contains many powerful cardiotonic drugs. In addition fruit rinds were dried in shade for 3-4 weeks and powdered. to the well documented folk uses of the plant in indigenous system Precisely weighed powdered samples (40g) were subjected to of medicine [3-5], extensive pharmacological assays have been successive soxhlet extraction using 350 ml each of Petroleum Ether conducted by many researchers using peruvoside and related (PE, 60-80°C), Chloroform (CH), Ethyl Acetate (EA) and Methanol glycosides on failing human heart and experimental models, which (MT) for 15-18 h in a soxhlet extractor. The fractions were dried, showed better cardiotonic activity [6-7]. Naturally occurring weighed and kept in labeled vials at 4ºC for further analyses. digitalis-like glycosides continues to be isolated from different plant Solvents and routine chemicals were procured from Merck and animal species; though digoxin and its acetyl and methyl Chemicals, Mumbai. derivatives are the cardiac glycosides most currently used in Preliminary phytochemical screening was conducted systematically therapeutics [2]. Recent researches revealed promising role of as per the standard protocol [19]. All four fractions (PE, CH, EA, MT) cardiac glycosides in the treatment of anticancer therapy [8-10]. from three color variants were analysed for the presence of primary Their novel role in healing several other diseases have also been and secondary metabolites like carbohydrates, proteins, starch, already established [11-13]. Most of the Thevetia glycosides has alkaloids, terpenoids, phenolics, steroids, saponins, cardiac been isolated and identified from different parts of the plant glycosides and anthraquinons. including fresh uncrushed leaves, seeds, flowers and stem bark [14- 18]. The plant commonly called ‘yellow oleander’ is cultivated as an Biological reference material, Peruvoside isolated from Thevetia ornamental for its attractive, light scented flowers in yellow, orange neriifolia was procured from Sigma- Aldrich, USA as 100 mg pack and white forms. These color variants are considered as three (CAS Number 1182-87-2) of 90% purity. All twelve samples (four morphoforms. Mature seeds are protected by fleshy mesocarp and extracts each from three plants) and reference drug were loaded hard stony endocarp. The green colored outer pulpy fruit wall (rind) side by side on TLC plates and from repeated experimental trails

Nesy et al. Int J Pharm Pharm Sci, Vol 6, Issue 8, 412-415 separation of various cardiac glycosides was achieved on chloroform cardiac glycosides; but peruvoside was observed as deep yellow methanol (8:2) mobile phase. Presence of peruvoside and other colored band with spraying reagent concentrated H2SO 4 [25] in members of cardiac glycoside family were detected using spraying chloroform and ethyl acetate fractions. These positive samples were reagent sulfuric acid [20]. Quantification of peruvoside and other further analysed via HPTLC fingerprinting for the simultaneous cardiotonic steroids was done by HPTLC fingerprinting using Camag separation and estimation of various cardiac glycosides and HPTLC system (Germany). Samples of CH and EA fractions from the quantification of peruvoside using biological reference material. three variants were prepared by dissolving 10mg of extract in 1 ml Extensive scientific research on this unattended fruit wall exposed of corresponding solvent, while 1mg/ml dilution was made for the occurrence of enormous biological potential in the form of reference compound. Sample solutions were centrifuged at 3000 cardiac glycosides and other phytochemicals. rpm for 3 min and spotted (5 µl) as bands of 6 mm width in Densitogram of HPTLC revealed that chloroform extract of yellow precoated silica gel aluminum plates 60 F254 (E Merck KGaA). Camag twin-trough developing chamber was saturated with 10 ml and white variants (YRCH & WRCH) showed the presence of 9 types chloroform: methanol (8:2) mixture mobile phase for 10 min at of cardiac glycosides as 9 peaks with different Rf values ranging room temperature. The developed chromatogram was dried at 80ºC from 0.03 to 1.00. Lesser diversity was observed in ORCH with 6 for 3 min and scanned using CAMAG TLC Scanner-3 at a speed of 20 compounds (Table 1). Comparison of compound 1 in all three CH mm/s at 220 nm. samples with respect to the Rf value and peak area, it is understood that this compound is identical in all three color variants. Higher Plates were visualized and documented using digital documentation percentage in ORCH may be due to the fact that it does not have system in a visualizer under UV light at 254 nm, 366 nm and white undergone hydrolysis and formed compound 2 as observed in YR light. Post chromatographic derivatization was carried out by and WR. Compound 3 was observed in WR only, because it may be spraying visualizing agent- Liebermann Burchard reagent onto the another hydrolysis product of compound 1. Almost similar Rf values plate. The plate was dried again at 120ºC for 20 minutes and images were detected for compound 4 in all samples, but minor quantity of were captured. From the recorded data, quantity of peruvoside was compound 5 was present in YR only. Subsequently eluted compound calculated in percentages and comparative table of other cardiac (6) in YR and WR illustrated quite similarity to reference drug and glycoside series was prepared. Six unknown samples and reference the elution time in WR prolonged further at the tail end when compound were chromatographed side by side on the same plate; compared to reference compound. Peruvoside purchased from and fingerprints were compared with respect to number, sequence, Sigma showed an elution phase from 0.63 to 0.74 (Fig 2A). Samples position and color of separated zones [21]. from all three variants possessed similar compound with elution period 0.63 to 0.75 with a ±0.03 variation. Minor variations can be RESULTS AND DISCUSSION neglected as all compounds in the same developing plate show this Qualitative phytochemical screening for the presence of diverse deviation. Similar Rf values showed by the remaining three groups of primary and secondary metabolites was carried out in all compounds (7, 8 & 9) revealed the relationship of Thevetia four fractions (PE, CH, EA & MT) of three studied morphovariant glycosides in nature structurally and functionally. Of course, all these plants. The results revealed the presence of carbohydrates, cardiotonic steroids have similar function. All these derivatives are alkaloids, flavonoids, steroids, saponins, terpenoids, and cardiac formed from the parent compound Thevetin [26]. Fractionation and glycosides in various fractions. All these classes of phytochemicals hydrolysis of Thevetin leads to the formation of a variety of have a wide range of bioactivities, so the plant is proved to be a compounds with interconvertable and rearrangable functional valuable reservoir of several compounds with immense therapeutic groups and other molecules. In chloroform extract, peruvoside was value [22-24]. TLC analysis was employed to detect the presence of quantified as the third major compound in all studied variants, and various cardiac glycosides, since the study was focused on these all investigated samples showed nearly the same fingerprint (Fig 2 metabolites especially peruvoside. In silica gel plates the Rf B-D). Usually, different developmental stages of plants or different (retardation factor) value of peruvoside was found as 0.63-0.65 plant organs may vary considerably in their qualitative and when chloroform: methanol (8:2) mixture was used as mobile phase. quantitative phytochemical composition. This difference in For all samples analysed, positive results were obtained for CH and constituent profile of the plant material may be determined by EA fractions, while PE and MT fractions showed negative result for several environmental factors (temperature, light, rain, soil pH) peruvoside. Appearance of purplish brown and blue green bands harvesting time, drying methods, extraction methods, duration, was considered as positive evidence for the presence of various enzymatic reaction during storage etc. [21].

Table 1: Comparative evaluation of Rf values of Cardiac Glycosides present in CH fraction of three color variants of Thevetia neriifolia fruit rind. YRCH (9) ORCH(6) WRCH(9) Peak no. Start Max End Area Start Max End Area Start Max End Area Rf Rf Rf % Rf Rf Rf % Rf Rf Rf % 1 0.03 0.05 0.10 8.96 0.03 0.05 0.08 10.48 0.01 0.05 0.08 7.05 2 0.23 0.26 0. 32 3.09 - - - - 0.23 0.25 0.26 0.89 3 ------0.33 0.36 0.39 1.91 4 0.40 0.45 0.50 10.06 0.41 0.44 0.49 4.15 0.40 0.44 0.49 10.26 5 0.51 0.52 0.54 0.92 ------6 0.58 0.62 0.64 3.50 - - - - 0.59 0.65 0.67 5.48 7 0.65 0.71 0.74 14.80 0.63 0.71 0.73 16.95 0.68 0.70 0.75 7.94 8 0.74 0.77 0.80 8.51 0.74 0.76 0.80 9.66 0.75 0.76 0.78 0.79 9 0.81 0.86 0.91 36.25 0.80 0.86 0.88 25.12 0.79 0.85 0.87 32.09 10 0.91 0.93 1.00 13.92 0.88 0.93 1.00 33.64 0.87 0.90 0.98 32.71 YRCH, ORCH, WRCH – CH fraction of Rind from Yellow, Orange and White variants (Compounds having similar Rf values are considered as same and tabulated in single row)

Fingerprint analysis of EA fraction of fruit rind (Fig E-G) is tabulated acetate than chloroform. Similarly, Compound 2 and 12 appeared as in Table 2. Compound 1, extracted by EA (Table 2) with elution another chief glycoside in all three samples. Minute quantities of range of 0.1 to 0.7 showed the highest area of 35-38 %, and can be some analytes (3, 6, 10, 14) were detected in all samples, where as a considered as identical to compound 1 present in CH fraction when few (4, 5, 7, 8, 9, 11, 13) were present in particular samples only. It Rf value was considered. This compound was more soluble in ethyl was noticed that moderate amount of peruvoside was observed in

413 Nesy et al. Int J Pharm Pharm Sci, Vol 6, Issue 8, 412-415 all three samples (compound 10) and quantity varied in the one way or the other. The complex molecules have a tendency of following order - yellow> orange > white. In white variant, a total of biotransformation into different derivatives due to the action of three compounds (Rf 0.59 to 0.77) were separated within the Rf endogenous enzymes [27]. Definitely, this genus contains many range of reference compound (Rf 0.63 to 0.74). Each morphovariant known and unknown cardiac glycosides as complex mixtures that plant contains more than a dozen similar but different compounds in occur together in the same plant.

Table 2: Comparative evaluation of Cardiac Glycosides present in EA fraction of three color variants of T. neriifolia fruit rind Yrea (9) Orea (12) Wrea (11) Peak No. Start Rf Max Rf End Rf Area % Start Rf Max Rf End Rf Area % Start Rf Max Rf End Rf Area % 1 0.03 0.05 0.07 37.72 0.01 0.05 0.07 36.64 0.02 0.04 0.07 35.16 2 0.08 0.10 0.14 25.70 0.07 0.10 0.13 24.99 0.07 0.08 0.11 10.28 3 0.15 0.18 0.20 2.08 0.13 0.16 0.18 4.12 0.11 0.12 0.14 1.16 4 0.20 0.22 0.25 1.28 - - - - 0.20 0.25 0.30 5.24 5 - - - - 0.36 0.38 0.41 0.98 - - - - 6 0.42 0.45 0.50 2.42 0.41 0.45 0.47 2.92 0.42 0.45 0.46 1.50 7 ------0.54 0.56 0.58 4.84 8 - - - - 0.58 0.61 0.63 3.81 0.59 0.62 0.66 6.59 9 - - - - 0.68 0.68 0.69 1.12 0.66 0.69 0.72 5.98 10 0.69 0.73 0.76 4.31 0.70 0.71 0.75 3.79 0.72 0.74 0.77 3.15 11 0.76 0.77 0.79 0.90 0.75 0.76 0.79 1.15 - - - - 12 0.83 0.88 0.95 23.75 0.83 0.88 0.92 16.93 0.83 0.88 0.95 25.18 13 - - - - 0.93 0.94 0.96 2.51 - - - - 14 0.96 0.98 1.00 1.83 0.97 0.98 1.00 1.03 0.98 0.99 0.99 0.91 YREA, OREA, WREA – EA fraction of Rind from Yellow, Orange and White variants

Results presented in Table 3 revealed that all morphovariant plants The description of the chromatogram prior and subsequent contained almost same amount of peruvoside. Higher amount can be derivatization with Liebermann Burchard reagent was presented in extracted using chloroform than ethyl acetate. Fig 1. The chromatogram of the reference compound (track 1-3) exhibits a prominent band in derivatized plate which can be Two similar compounds having Rf values within the range of visualized at both lower (254 nm) and higher (366 nm) standard values are considered as same and may be formed by the wavelengths. Likewise, eluted compounds with identical nature can rearrangement of functional groups that could occur due to various be recognized by looking its position and band color in samples. reasons. Detailed studies are needed for each component to prove the Bands were narrower, but visible in CH (track 4-6) and EA (track 7- structural similarities or variations, if any. Similar analogues were 9) samples in the chromatogram against the sharp and prominent observed in our earlier studies with seed and flower extracts [28-29]. ones of standard compound.

Table 3: Quantification of Peruvoside in CH and EA fractions of fruit wall of three color variant plants Sample Start Rf Max Rf End Rf Area % Area % of peru Total Peru 0.63 0.67 0.74 57.8 15544.4 - - YRCH 0.65 0.71 0.74 14.8 4696.1 2.72 3.26* ORCH 0.63 0.71 0.73 16.95 4558.5 2.64 WRCH 0.59 0.65 0.67 5.48 1319.0 0.76 ,, 0.68 0.70 0.75 7.94 1908.6 1.11 3.24** YREA 0.69 0.73 0.76 4.31 925.1 0.54 OREA 0.68 0.68 0.69 1.12 236.9 0.14 ,, 0.70 0.71 0.75 3.79 801.8 0.46 3.07*** WREA 0.59 0.62 0.66 6.59 1091.9 0.63 ,, 0.66 0.69 0.72 5.98 990.6 0.57 Total Peruvoside present in *Yellow, ** Orange, ***White variants

A-C HPTLC fingerprints of T. neriifolia fruit wall extracts of yellow, orange and white forms before derivatization under 254 nm, 366 nm wavelength and day light (Tracks 1-3 Standard drug peruvoside; 4-6 chloroform fractions; 7-9 ethyl acetate fractions). D-F HPTLC profile after derivatization with Liebermann Burchard Reagent, photo documentation under 254 nm, 366 nm wavelength and day light. Cardiac glycosides are a group of natural products with specific action on myocardial contraction. In large dozes, they are toxic and bring about cardiac arrest but in lower doses, they are important drugs in the treatment of congestive heart failure and associated atrial fibrillation [30]. Use of cardiac glycosides of biological origin in treating failing heart was widely accepted from nineteenth century. Peruvoside possess a very good cardiotonic activity and Thevetia glycosides (Peruvoside, Theveneriin, Neriifolin) can be converted to digitoxigenin and digitoxose

by enzymatic reaction and by selective 1, 2-beta hydroxylation to yield Fig. 1: Chromatographic plate of standard drug Peruvoside and digoxin [31]. Huge quantities of cardiac glycosides available in other cardiac glycosides of T. neriifolia fruit wall extracts before decaying fruit wall can thus be converted into commercial products and after derivatization. for valuable cardiotonics.

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